Direct conversion of adult human fibroblasts into functional endothelial cells using defined factors
Direct conversion of adult human fibroblasts into functional endothelial cells using defined factors
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DOI：
10.1016/j.biomaterials.2021.120781
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发表时间：
2021-04-10
期刊：
影响因子：
14
通讯作者：
Kim, Hyo-Soo
中科院分区：
文献类型：
--
作者：
Han, Jung-Kyu;Shin, Youngchul;Kim, Hyo-Soo
我们的目的是直接将成人皮肤成纤维细胞（aHDF）转化为功能性内皮细胞（EC）。构建了编码内皮转录因子（TF）的慢病毒载体。我们检查了用于产生小鼠诱导的EC（iEC）的五种TF（FOXO 1、ER 71、KLF 2、TAL 1和LMO 2）是否可以将aHDF转化为人iEC。28天后转导慢病毒结构，32.1？5.1%的细胞表达血管内皮细胞钙粘蛋白（VE-cadherin）。因子筛选显示，只有三个因子（3F：ER 71，KLF 2和TAL 1）是诱导VE-钙粘蛋白（+）细胞所必需的（49.4？3.5%）。然而，全转录组测序显示VE-钙粘蛋白（+）细胞没有完全重编程。成熟的IEC双阳性VE-钙粘蛋白/Pecam 1（DP细胞）与鹅卵石外观获得的频率只有5.1？百分之零点六。利用全转录组分析，筛选可能阻断转化的潜在因素。在候选者中，TWIST 1敲低提高了转化效率。EMT抑制剂罗格列酮也能提高转化效率。此外，第二个第二阶段的转化过程，其中VE-钙粘蛋白（+）细胞再孵育两周，进一步提高了效率。6周的最终方案产生的转化率为19.6？3.0% iEC，由DP细胞定义，描述了各种分析中成熟EC的性质。进一步的分析表明，iECs的遗传和表观遗传特征与功能性EC相似。总的来说，aHDF可以通过ER 71、KLF 2和TAL 1的转导，结合两种EMT抑制剂（siTWIST 1和罗格列酮），然后进行第二阶段转化而转化为功能性EC。
We aimed to directly convert adult human dermal fibroblasts (aHDFs) into functional endothelial cells (ECs). Lentiviral vectors encoding endothelial transcription factors (TFs) were constructed. We examined whether five TFs (FOXO1, ER71, KLF2, TAL1, and LMO2) used for the generation of mouse induced ECs (iECs) could convert the aHDFs into human iECs. Twenty-eight days after transduction with lentiviral constructs, 32.1 ? 5.1% cells expressed vascular endothelial (VE)-cadherin. Factor screening revealed that only three factors (3F: ER71, KLF2, and TAL1) were necessary to induce VE-cadherin (+) cells (49.4 ? 3.5%). However, whole transcriptome sequencing showed that VE-cadherin (+) cells were not completely reprogrammed. Mature iECs double-positive for VE-cadherin/Pecam1 (DP cells) with a cobblestone appearance were obtained at a frequency of only 5.1 ? 0.6%. Using whole transcriptome analysis, the potential factors that could block the conversion were screened. Among candidates TWIST1-knockdown enhanced efficiency of conversion. Rosiglitazone, an inhibitor of epithelial-mesenchymal tran-sition (EMT), also improved the conversion efficiency. Moreover, a 2nd second-stage conversion process, in which VE-cadherin (+) cells were incubated for addi-tional two weeks, further enhanced the efficiency. The final protocol for 6 weeks yielded a conversion rate of 19.6 ? 3.0% iECs, defined by DP cells depicting the nature of mature ECs in various analyses. Further analyses revealed that the genetic and epigenetic profiles of iECs resembled those of functional ECs. Collectively, aHDFs can be converted into functional ECs through the transduction of ER71, KLF2, and TAL1, combined with two EMT inhibitors (siTWIST1 and rosiglitazone), followed by 2nd stage conversion.